J. Douglas Waterfield

Proinflammatory Cytokine Profiles in Pulp Fibroblasts Stimulated with Lipopolysaccharide and Methyl Mercaptan

Pulpal disease is intimately associated with the immune systems response to bacteria products. Clinical pathology is mediated in part by the production of pyrogenic cytokines, especially interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and IL-6. Methyl mercaptan (CH3SH), a volatile sulfur compound produced by anaerobic Gram-negative bacteria, has been shown to contribute to the production of IL-1 by human mononuclear cells. In this report, we investigated the production of IL-1, TNF-alpha, and IL-6 by human pulp fibroblasts when stimulated for various periods of time with lipopolysaccharide (LPS) with or without the presence of CH3SH. We found that LPS and CH3SH had no effect on the production of IL-1 or TNF-alpha. However, LPS stimulated IL-6 production, and this production was augmented when CH3SH was present. We conclude that the volatile sulfur compound CH3SH plays a role in activation and modulation of the immune response through its role in production of IL-6.

The effect of surface roughness on RAW 264.7 macrophage phenotype.

Monocyte-derived cells, including macrophages and foreign body giant cells, can determine the performance of implanted devices. Upon contact with biomaterials, macrophages can be activated into a classic inflammatory (M1) or wound-healing (M2) phenotype. Previously, we showed that high macrophage density on rough SLA implants was associated with early bone formation. This study examined a possible mechanism, namely, surface roughness activation of macrophages to the M2 phenotype to enhance bone formation on the SLA surface. RAW 264.7 macrophages were seeded on SLA or smooth (Po) epoxy substrates and the expression of the M1 and M2 specific markers, NOS2 and Arg-1 measured by qPCR on days 1, 3, and 5. Additionally, secretion of inflammation-associated cytokines and chemokines was studied by antibody arrays and ELISAs. Controls included RAW 264.7 macrophages primed into the M1 or M2 phenotypes by LPS/IFN-γ and IL-4, respectively. Rough SLA surfaces did not activate Arg-1 and NOS2 expression, but relative to Po surfaces MCP-1 and MIP-1α were upregulated after 5 days, whereas the secretion of the M1-associated chemokine IP-10 was lowered. RAW 264.7 macrophages on the SLA surface thus adopted elements of an M2-like phenotype, suggesting that when implanted the SLA surfaces may enhance wound repair.

Stimulation of enzyme and cytokine production by methyl mercaptan in human gingival fibroblast and monocyte cell cultures

The volatile sulphur compound methyl mercaptan (CH3SH) is a by-product of protein metabolism and a principal component of oral malodour. This investigation examines the effect of CH3SH on the enzymatic activities of cathepsins B and G and elastase, and on the production by human gingival fibroblasts of two key factors, prostaglandin E (PGE) and cAMP, of the PGE2-cAMP-dependent pathway, which may contribute to the increased production of collagenase and tissue destruction in human periodontal disease. The results demonstrate that CH3SH alone, or in combination with interleukin-1 (IL-1) or lipopolysaccharide, can significantly enhance the secretion of PGE2, cAMP and procollagenase by human gingival fibroblasts. CH3SH also stimulated mononuclear cells to produce IL-1, which can increase cAMP production, and act in synergism with the direct effect of CH3SH on cAMP. CH3SH also significantly enhanced the activity of cathepsin B, moderately suppressed that of cathepsin G, but did not significantly affect elastase. These results provide evidence that CH3SH could be a contributing factor in the enzymatic and immunological cascade of events leading to tissue degradation in periodontal diseases.

The CCL3 Family of Chemokines and Innate Immunity Cooperate In Vivo in the Eradication of an Established Lymphoma Xenograft by Rituximab

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these β-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11–70), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2–3 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11–70) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2−/−cγ−/− mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces β-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor.

Uptake of verteporfin by articular tissues following systemic and intra-articular administration.

Photodynamic therapy (PDT) using the photosensitizer BPD‐Verteporfin (liposomal benzoporphyrin derivative‐monoacid ring A) has been shown in previous studies to be effective in the amelioration of inflammatory arthritis in both the MRL‐lpr mouse and the New Zealand White (NZW) rabbit models, and could potentially offer alleviation of certain inflammation‐related symptoms of rheumatoid arthritis. Time and dose dependency of BPD‐MA tissue uptake was carried out in the inflamed synovium and other articular and peri‐articular tissues following intravenous and intra‐articular administration in the NZW rabbit model. As some articular and peri‐articular tissues are difficult to extract, this study uses a rapid fluorimetric sampling of tissues following dissolution in Soluene 350. Our results showed that i.v. injected BPD‐MA preferentially distributed in the inflamed synovium, and in tissues with a high degree of vascularization. Little or no association was found with avascular tissues such as cartilage and tendons. Clearance from the synovium was rapid, supporting earlier rather than late light treatment. Much higher association of BPD‐MA with the synovium was achieved using intra‐articular injection, and BPD‐MA concentrations were maintained at relatively steady levels for several hours. These observations support the possibility that PDT could offer a safe, highly versatile clinical option for the management of inflamed joints in autoimmune disorders.

The effect of surface topography on early NFκB signaling in macrophages

Surface topography modulates macrophage expression of pro-inflammatory cytokines through triggering of a number of different signaling pathways. In this article, we investigated the early activation of the NFκB pathway in RAW 264.7 macrophages in response to four surface topographies: mechanically polished (PO), coarse sand blasted (CB), acid etched (AE), and sandblasted and acid etched (SLA). We found that activation of the NFκB pathway was topography dependent. The PO and CB surfaces showed the highest level of activation, followed by the AE, then the SLA. Addition of suboptimal stimulatory concentrations of lipopolysaccharide (LPS) enhanced the response. Second, we determined that topography dependent cell signaling occurred in the absence of fetal bovine sera in the media. Third, we demonstrated that disruption of the lipid rafts by removal of cholesterol from cells in suspension using methyl β cyclodextrin (MβCD) affected signaling through the NFκB pathway and transcription of the pro-inflammatory cytokine IL-1 β, but did not affect cell adhesion, spreading or morphology. The number of macrophages adhered to the surfaces after 30 min followed the order PO, CB, AE, and SLA. In conclusion, our study suggests that one mechanism by which surface topography modulates activation of the NFκB pathway is through cholesterol-enriched raft-associated adhesive/signaling structures.

The effect of surface topography on cell shape and early ERK1/2 signaling in macrophages; linkage with FAK and Src†

Implant surface topography can modulate macrophage behavior during wound healing by the production of proinflammatory cytokines. This study investigated the activation of FAK, Src, and ERK1/2 signaling intermediates of the proinflammatory ERK1/2 pathway in RAW 264.7 macrophages in response to polished (P), coarse-grit-blasted (B), acid etched (E), and grit-blasted and etched (SLA) surface topographies. In addition, the effects of these topographies on cell spreading, vinculin organization, and viability were determined. Macrophages on the SLA surface changed from predominantly well-spread cells to ones with a more spherical morphology over time. In contrast, macrophages on the P surface changed from being predominantly spherical cells to well spread. The morphological changes were associated with changes in the distribution of vinculin. The overall patterns of the pFAK, pSrc, pERK1/2 levels as well as pERK1/2 nuclear translocation associated with cell shape with greater activation being seen with a more spread morphology. These results suggest that surface topography differentially activates signaling pathways that affect cell function and raise the possibility that topographies can be designed to optimize desired cell responses.

Photodynamic therapy in immune (non-oncological) disorders: focus on benzoporphyrin derivatives.

This review examines the efficacy of photodynamic therapy in the treatment of immunological disorders. Photodynamic therapy (PDT) is a 2-step procedure. Firstly, a photosensitiser is introduced into the body, where it accumulates selectively in cells with elevated metabolism, such as cancer cells or activated cells of the immune system. Second, light is applied at a wavelength that excites the photosensitiser, producing a variety of short-lived oxygen-derived species. The effect is dependent on the doses of both photosensitiser and activating light. The mechanisms of action of PDT are multifactorial. Induction of high levels of oxidative stress results in necrotic cell death, while lower intensity oxidative stress initiates apoptosis. Sublethal doses may result in the modification of cell surface receptor expression levels and cytokine release and consequently influence cell behaviour. Immunomodulatory PDT (IPDT) utilises mainly apoptotic and sublethal doses.The studies reported here utilise verteporfin, a benzoporphyrin-derived chlorinlike photosensitiser. Veteporfin is a second generation photosensitiser, displaying rapid clearance and consequently a reduced period of skin photosensitivity compared with the first generation photosensitiser, porfimer sodium. In vivo studies showed that IPDT was effective in alleviating immunopathology in murine models of arthritis, contact hypersensitivity, experimental allergic encephalomyelitis and retention of allogeneic skin grafts. Based on these findings, early stage clinical trials with IPDT were initiated recently for the treatment of psoriasis, psoriatic arthritis and rheumatoid arthritis.While verteporfin has been the photosensitiser which pioneered IPDT, a new benzoporphyrin derivative photosensitiser, QLT0074, is under development. This has demonstrated an enhanced avidity for target cells as well as improved clearance characteristics.

Evaluation of the Immunotoxicity of Benzoporphyrin Derivative (Bpd-Ma) in Mice

Photodynamic therapy has been shown to selectively eliminate activated lymphocytes in a number of experimental situations. These findings have important implications in therapies involving selective immunomodulation. In this study we report the effects of intravenous dosing with the photosensitizer benzoporphyrin derivative-monoacid A(BPD) on normal immunological function. Therapeutic doses of BPD and light had no effect on natural killer cell activity, the mixed lymphocyte reaction, cell-mediated lympholysis, the primary immune response to sheep red blood cells, or the secondary memory response to T cell-dependent antigens. In non-light treated controls, BPD at concentrations up to 10-fold higher had a limited effect on cell-mediated lympholysis. We conclude that the primary effect of BPD in several therapeutic modalities in not due to a generalized suppression of the immune system.

Novel grooved substrata stimulate macrophage fusion, CCL2 and MMP-9 secretion

Rough surface topographies on implants attract macrophages but the influence of topography on macrophage fusion to produce multinucleated giant cells (MGC) and foreign body giant cells (FBGC) is unclear. Two rough novel grooved substrata, G1 and G2, fabricated by anisotropic etching of Silicon <110> crystals without the use of photolithographic patterning, and a control smooth surface (Pol) were produced and replicated in epoxy. The surfaces were compared for their effects on RAW264.7 macrophage morphology, gene expression, cyto/chemokine secretion, and fusion for one and five days. Macrophages on grooved surfaces exhibited an elongated morphology similar to M2 macrophages and increased cell alignment with surface directionality, roughness and cell culture time. Up-regulated expression of macrophage chemoattractants at gene and protein level was observed on both grooved surfaces relative to Pol. Grooved surfaces showed time-dependent increase in soluble mediators involved in cell fusion, CCL2 and MMP-9, and an increased proportion of multinucleated cells at Day 5. Collectively, this study demonstrated that a rough surface with surface directionality produced changes in macrophage shape and macrophage attractant chemokines and soluble mediators involved in cell fusion. These in vitro results suggest a possible explanation for the observed accumulation of macrophages and MGCs on rough surfaced implants in vivo.